September 2008
Volume 49, Issue 9
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Cornea  |   September 2008
A Rapid Separation of Two Distinct Populations of Mouse Corneal Epithelial Cells with Limbal Stem Cell Characteristics by Centrifugation on Percoll Gradient
Author Affiliations
  • Magdalena Krulova
    From the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; the
    Faculty of Natural Sciences, Charles University, Prague, Czech Republic; the
  • Katerina Pokorna
    From the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; the
    Faculty of Natural Sciences, Charles University, Prague, Czech Republic; the
  • Anna Lencova
    From the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; the
    Eye Clinic Lexum, Prague, Czech Republic; and the
  • Jan Fric
    From the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; the
    Faculty of Natural Sciences, Charles University, Prague, Czech Republic; the
  • Alena Zajicova
    From the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; the
  • Martin Filipec
    Eye Clinic Lexum, Prague, Czech Republic; and the
  • John V. Forrester
    Department of Ophthalmology, University of Aberdeen, Aberdeen, Scotland.
  • Vladimir Holan
    From the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; the
    Faculty of Natural Sciences, Charles University, Prague, Czech Republic; the
Investigative Ophthalmology & Visual Science September 2008, Vol.49, 3903-3908. doi:10.1167/iovs.08-1987
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      Magdalena Krulova, Katerina Pokorna, Anna Lencova, Jan Fric, Alena Zajicova, Martin Filipec, John V. Forrester, Vladimir Holan; A Rapid Separation of Two Distinct Populations of Mouse Corneal Epithelial Cells with Limbal Stem Cell Characteristics by Centrifugation on Percoll Gradient. Invest. Ophthalmol. Vis. Sci. 2008;49(9):3903-3908. doi: 10.1167/iovs.08-1987.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To detect and isolate cells with stem cell (SC) characteristics in the limbus of the mouse.

methods. Limbal tissues from BALB/c mice were trypsin-dissociated and separated on the gradient Percoll (Fluka, Buchs, Switzerland). Several fractions were isolated and characterized by real-time PCR for the presence of limbal SC markers and differentiation markers of corneal epithelial cells by flow cytometry for the determination of the side-population (SP) phenotype and growth properties in vitro.

results. Cells retained in the lightest fraction (40% Percoll) and in the densest fraction (80% Percoll) of the gradient were both enriched for populations with a high expression of the SC markers ABCG2 and Lgr5 and also expressing the SP phenotype. However, the lightest fraction (representing approximately 12% of total limbal cells) contained cells with the strongest spontaneous proliferative capacity and expressed the corneal epithelial differentiation marker K12. In contrast the densest fraction (<7% of original cells) was K12 negative and contained small nonspontaneously proliferating cells, which instead were positive for p63. Unexpectedly, cells from this fraction had the highest proliferative activity when cultured on a 3T3 feeder cell monolayer.

conclusions. These findings demonstrate the presence of two distinct populations of corneal epithelial cells with limbal SC characteristics, based on differential expression of the keratin-specific marker K12 and transcription factor p63, and suggest a difference in developmental stage of the two populations, with the K12p63+ population being closer to the primitive limbal SC.

The corneal surface is renewed under normal physiological conditions or during healing after injury by cells that migrate from the limbus. These cells originate from limbal stem cells (SCs) which reside in the basal layer of the limbus and represent a minor fraction of a heterogeneous limbal cell population. 1 2 3 When the limbal SC population is damaged or depleted (e.g., after injuries such as alkaline burns or genetically as in the Pax6 heterozygotic mouse), 4 5 healing of the corneal surface is prevented, and the cornea is invaded by cells from conjunctival epithelia resulting in poor epithelialization, vascularization, and corneal scarring, potentially leading to blindness. In such cases, transplantation of limbal SCs may be the only way to treat various eye surface injuries or diseases. 6 7 8  
To study the biological properties and to provide a potential source of limbal SCs, methods are needed to isolate or at least enrich limbal SCs from the heterogenous population of limbal cells. The absence of a definitive biological or phenotypic marker contributes a degree of uncertainty to the unequivocal isolation and characterization of limbal SCs. So far, a variety of SC markers have been proposed to identify this population of cells. Among the major characteristics proposed for SCs are small size 9 10 ; slow-cycling properties 11 ; the expression of intracellular markers such as drug resistance transporter ABCG2, the transcription factor p63, the integrin α9, and the cytokeratin K19 12 13 14 15 16 17 18 ; and the absence of corneal differentiation markers K3 and K12 or connexin 43. 1 19 20 21 Recently, the leucine-rich-repeat-containing G-protein-coupled receptor, Lgr5, has been suggested to mark of SCs in multiple adult tissues. 22 SCs express the side-population (SP) phenotype based on the ability to efflux the DNA-binding dye Hoechst 33342. 18 23 Although SCs are in vivo in a quiescent state and are only slowly dividing, in vitro they possess the highest colony-forming unit efficacy and growth properties on feeder cell monolayers. 10 24 25 Some of these characteristics, mainly the SP phenotype and small cell size, have been used in attempts to isolate SCs from human and rabbit limbuses or limbal cell cultures. 10 12 24 Another approach to enriching human limbal epithelial cells with SC properties is based on their differential adhesiveness to collagen type IV. 26 To date, probably due to the small size of the mouse eye, no attempt to isolate and characterize limbal SCs in the mouse has been reported. We show here for the first time that two distinct populations of corneal epithelial cells with limbal SC characteristics can be isolated from the mouse limbus by centrifugation on a Percoll gradient (Fluka, Buchs, Switzerland). These cells share characteristics of SCs with human or rabbit limbal SCs and can be used for the study of limbal SC properties and for studies of limbal SC deficiency in experimental mouse models such as the Pax6+/− mouse. 
Material and Methods
Mice
Mice of the inbred strain BALB/c of both sexes at the age of 2 to 4 months were used in these experiments. The animals were obtained from the breeding unit of the Institute of Molecular Genetics (Prague, Czech Republic). The use of animals was approved by a local Ethics Committee of the Institute of Molecular Genetics, and all animals were handled in full accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Isolation of Limbal Cells
Limbal tissue was obtained by scissor dissection of the eyes of killed mice guided by an operating microscope. Limbal tissues from 10 to 12 BALB/c mice were pooled and cut into small pieces in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO). The tissue was centrifuged (8 minutes at 250g), and the pellet was subjected to digestion with trypsin from porcine pancreas (Sigma-Aldrich). The procedure consisted in 10 trypsinization cycles (300 μL of 0.5% trypsin solution per 10 limbuses, 10 minutes incubation in 37°C). The supernatants (tissue-free solution) from each trypsinization step were harvested into an excess (30 mL) of RPMI 1640 medium with 10% fetal calf serum (FCS; Sigma-Aldrich) on ice, and the trypsinization procedure was repeated on the residual pellet. After the last trypsinization step, the harvested cell suspension was filtrated through a nylon mesh and centrifuged for 8 minutes at 250g. The pellet was resuspended in 1.2 mL of RPMI 1640 medium, and the number of cells was determined by hemocytometry. 
Percoll Gradient Centrifugation
To prepare a stock solution, nine parts Percoll was mixed with one part 10× concentrated phosphate buffered saline (PBS). From the stock solution, a 40%, 50%, 60%, 70%, or 80% Percoll solution was prepared by dilution in 1× PBS. A Percoll gradient was prepared in a 10-mL test tube by overlaying of 1.0 mL of each Percoll dilution 80% through 40%. Finally, 1.0 mL of suspension of trypsin-dissociated limbal cells was gently overlaid on the top of the Percoll gradient. The gradient was centrifuged for 10 minutes at 300g at 4°C. 
After centrifugation, the separated layers of cells on individual Percoll concentrations could be directly visualized, and individual cell layers (as well as the cell pellet) were harvested into RPMI 1640 medium with 5% of FCS and washed three times by centrifugation (8 minutes at 250g). After the last washing, the cells were resuspended in 500 μL of RPMI 1640 medium containing 10% of FCS, 10 mM HEPES buffer, antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin) and 5 × 10−5 M 2-mercaptoethanol (hereinafter called complete RPMI 1640 medium). The number of cells in each fraction was then determined. 
Quantitative Real-Time Polymerase Chain Reactions
The expression of genes for mouse ABCG2, Lgr5, the α isoform of DeltaNp63 (p63), and K12 was determined by quantitative real-time PCR. Total RNA was isolated from unseparated limbal cells or cells from individual Percoll fractions (TRI Reagent; Molecular Research Center, Cincinnati, OH). One microgram of total RNA was reverse transcribed into cDNA in 20-μL reaction mixture, as described previously. 27  
Quantitative real-time PCR was performed (iCycler; Bio-Rad, Hercules, CA), and the data were analyzed (iCycler Detection System, ver. 3.1; Bio-Rad). The specificity of the amplified products was checked by the melting analyses. Master mix (iQ SYBR Green Supermix; Bio-Rad) was used in all experiments. Each single experiment was performed in triplicate, and the reaction efficiency for each gene was estimated by the dilution curve method. The relative quantification model with efficiency correction was applied to calculate expression of target genes in comparison to GADPH, which was used as the housekeeping gene. The primers are described in Table 1 . The PCR parameters for 25-μL reactions included denaturation at 95°C for 3 minutes, then 40 cycles at 95°C for 10 seconds, annealing at 60°C for 20 seconds, and elongation at 72°C for 20 seconds. Fluorescence data were collected at each cycle after an elongation step at 80°C for 5 seconds. 
Light-Scattering Measurements
Trypsin-dissociated unseparated limbal cells or limbal cells from individual Percoll fractions were resuspended in PBS with 5% FCS and 2 μg/mL of propidium iodide (Sigma-Aldrich) for 20 minutes at 4°C. The light-scattering properties of the cells were measured in a flow cytometer (BD LSRII; BD Biosciences, Franklin Lakes, NY), with an Argon laser (488 nm) providing the probing beam, and the FSC/SSC density plots of viable cells were generated. 
Hoechst 33342 Exclusion Assay
Freshly isolated unseparated limbal cells or cells of individual fractions from a Percoll gradient were resuspended at a concentration of 1 × 106 cells/mL in Dulbecco;1 Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2% FCS and incubated with 5 μg/mL Hoechst 33342 (Sigma-Aldrich) dye. To determine the effect of verapamil on the Hoechst 33342 efflux, the cells were preincubated with verapamil (80 μM; Sigma-Aldrich) for 5 minutes before the addition of the Hoechst 33342 dye. After the incubation for 60 minutes at 37°C, propidium iodide (2 μg/mL) was added to exclude dead cells from the analysis, and the cells were then analyzed on a flow cytometer (FACSVantage SE; BD Biosciences), as described by Goodell et al. 28 Briefly, Hoechst 33342 was excited at 350 nm with a UV laser (Enterprise II-621; Coherent, Santa Clara, CA), and fluorescence emission was detected through 450-nm band-pass (Hoechst blue) and 660-nm long-pass (Hoechst red) filters. 
Spontaneous Proliferation of Limbal Cells In Vitro
Unseparated limbal cells or cells from individual fractions from the Percoll gradient were diluted to a concentration of 5 × 104 cells/mL in complete RPMI 1640 medium. One hundred microliters per well of cell suspension was incubated in triplicate in 96-well tissue culture plates (Nunc, Roskilde, Denmark). Cell proliferation was determined by adding 1 μCi/well of [3H]thymidine (Nuclear Research Institute, Rez, Czech Republic) for the last 8 hours of the 96-hour incubation period. The cells were harvested (Automasch 2000 harvester; Dynatech, Burlington, MA), and [3H]thymidine activity was determined. 
Cell Proliferation on Feeder 3T3 Fibroblasts
Irradiated (150 Gy) mouse 3T3 fibroblasts were seeded as feeder cells at a concentration of 104 cells/well in a volume of 50 μL of complete RPMI 1640 medium into wells of 96-well tissue culture plates (Nunc) and incubated overnight. Unseparated limbal cells or cells from individual Percoll gradient fractions (5 × 103 cells in 50 μL) were then added in triplicate into wells with feeder cells. The cultures were incubated for 96 hours, [3H]thymidine (1 μCi/well) was added for the last 8 hours of the incubation period, and the incorporated radioactivity was determined as just described. 
Statistical Analysis
The statistical significance of differences between individual groups was calculated by Student’s t-test. 
Results
Limbal Cell Isolation and Percoll Gradient Separation
Trypsin-dissociation of limbal tissue from one BALB/c mouse yielded on average 0.5 to 1 × 105 cells that were heterogeneous in both size and morphology, as determined by the light-scattering profile (Fig. 1) . Accordingly, limbal cells obtained by trypsin digestion (see the Methods section) from 10 BALB/c mice were pooled and separated on the Percoll gradient. The proportion of cells retained in individual Percoll gradient fractions and the recovery of original cells are shown in Table 2 . While the lightest fraction (40% Percoll) contained predominantly large and more heterogeneous cells with a smaller nucleus/cytoplasm ratio, the fraction retained on the 80% Percoll (densest fraction) was enriched in small dense cells with a higher ratio nucleus/cytoplasm. The pelleted fraction contained dead cells, cell debris, and fragments of corneal tissue and thus was not included in the analyses. 
Phenotype Characterization
The expression of genes for the putative SC markers ABCG2, Lgr5, and p63 and for corneal epithelial cell differentiation marker K12 was determined by using real-time PCR in unseparated limbal cells and limbal cell fractions isolated from Percoll gradient. As demonstrated in Figure 2 , both the lightest (40% Percoll) and densest (80% Percoll) fractions were enriched in cells expressing the SC markers ABCG2 and Lgr5, whereas the fraction from the middle region of the Percoll gradient had a lower expression of these markers compared to unseparated limbal cells. The marker of primitive and SCs p63 was expressed selectively in the dense (80% Percoll) cell fraction (Fig. 2) . The corneal differentiation marker K12 was expressed predominantly in larger cells retained on 40% or 50% Percoll and was absent in small cells of the dense fractions (Fig. 2)
Identification of the SP Phenotype on the Basis of the Efflux of Hoechst 33342 Dye
Using flow cytometry we first demonstrated that normal fresh mouse limbus contains a small population of cells (SP cells) that can be detected by verapamil-sensitive disappearance of a unique tail of a low Hoechst 33342 blue-red fluorescence (Figs. 3A 3B) . This population represented 2.3% to 5.4% of total mouse limbal cells. Analysis of individual fractions from the Percoll gradient showed that SP cells were enriched in light and dense fractions (40% and 80% Percoll) and were relatively decreased in cells from the intermediate fractions of the Percoll gradient (Figs. 3C 3D 3E 3F 3G 3H)
Forward-scattering analysis was performed to determine the relative cell size and granularity of SP cells from unseparated total limbal cells (Fig. 4A)or of the fractions from the 40% (Fig. 4B)and the 80% (Fig. 4C)Percoll gradients. The SP cells from the 40% gradient fraction were apparently more heterogeneous with respect to granularity than were the small and more uniform SP cells in the 80% fraction. 
Growth Properties of Limbal Cell Fractions
To evaluate the proliferative capacity of the various cell fractions, we seeded the cells at a concentration of 5 × 103 cells/well into 96-well tissue culture plates (in a volume of 100 μL complete RPMI 1640 medium/well). Cell proliferation was determined by incorporation of [3H]thymidine. As demonstrated in Figure 5A , the 40% Percoll gradient cell fraction proliferated with a significantly higher intensity than did the unseparated limbal cells, whereas cells from the 70% or 80% fractions had very limited proliferative activity. 
We also assessed the proliferative capacity of individual cell populations on irradiated 3T3 fibroblast feeder cells in 96-well tissue culture plates. The proliferative activity of limbal cells was determined according to the incorporation of radioactivity. The results are expressed as the ratio of the proliferative activity of limbal cells on a 3T3 feeder layer to the proliferation of the same cells in wells without feeder cells. As demonstrated in Figure 5B , in this experimental setting, the strongest proliferative activity was observed in the small cell population retained in the 70% and 80% Percoll gradient fractions, while the proliferative capacity of cells from the 40% and 50% fractions was not increased over spontaneous proliferation levels without feeder cells. 
Discussion
SCs for the renewal of the corneal surface epithelium are located in the basal layer of the limbal epithelium. These cells, which are characterized by small size; a low rate of replication; expression of certain markers such as a transporter ABCG2, p63, integrin α9, or K19; and by the expression of the SP phenotype have been described in human, 10 12 13 14 rabbit, 24 29 and rat 23 corneas. To date, there are no reports of isolation and characterization of limbal SCs in the mouse. 
We have shown that cells sharing morphologic, phenotypical, and functional characteristics with human and rabbit limbal SC can also be found in the mouse limbus. To dissociate the limbal tissue into a single cell suspension, we compared various enzymatic digestion protocols including dispase treatment, combination of dispase, and trypsin or trypsin digestion alone. Repeated short trypsin digestions were the optimal method, allowing recovery of 0.5 to 1 × 105 limbal cells per one BALB/c mouse. These cells were heterogeneous with respect to the size, granularity, and the ratio of cytoplasm to nucleus and could be separated into six subpopulations on a 40% to 80% discontinuous Percoll gradient. The densest fraction (80% Percoll), representing approximately 7% of original limbal cells, was enriched in cells showing morphologic characteristics described for the human and rabbit limbal SCs: small size, dense cells, and a low ratio of cytoplasm to nucleus. Further analysis of this fraction showed that this population was enriched in cells expressing the SP phenotype (>30% of SP cells in comparison to 2%–5% of cells expressing the SP phenotype in the whole limbus) and the fraction also had significantly enhanced expression of the SC markers ABCG2, Lgr5, and p63 in comparison with unseparated limbal cells. In addition, these cells were in a nonproliferative quiescent state as demonstrated by their very low spontaneous uptake of radioactive thymidine in vitro. However, when cultured on a feeder cell layer, they demonstrated considerable proliferative capacity. All these characteristics resemble properties of limbal SC described in human or rabbit limbal tissue. 12 13 14 24 29  
A second cell fraction showing at least some characteristics of limbal SCs was detected in the lightest cell population (40% Percoll gradient) and represented approximately 12% of the total limbal cell population. These cells expressed genes for the SC markers ABCG2 and Lgr5, and over 20% of the cells expressed the SP phenotype, a property of SCs. 30 31 However, unlike the dense cell population (80% gradient) which also expressed the SP phenotype and SC markers, the light cell population was positive for corneal differentiation marker K12 and had the highest spontaneous proliferative capacity (significantly higher than unseparated limbal cells) and their proliferation response did not increase when they were cultured on a feeder cell monolayer. Thus, two separable populations of mouse limbal cells which have SC characteristics (ABCG2 and Lgr5 expression, the SP phenotype) can be obtained by centrifugation on Percoll gradient. The cells retained in the intermediate layer of the gradient (60% Percoll) had lower expression levels of SC markers and a lower percentage of cells expressing the SP phenotype than original unseparated limbal cells. 
The results thus demonstrated that two distinct populations of limbal cells with SC characteristics can be isolated in the mouse. Both populations contained cells expressing the SP phenotype based on the efflux of Hoechst 33342 dye. However, forward-scattering analysis of SP cells from the top and bottom fractions showed that both populations differ in their size and granularity. The number of SP cells in the unseparated mouse limbus was 3.8% (average from five experiments) of total limbal cells, substantially higher than the number of slow-cycling corneal epithelial cells found at the mouse limbus 32 or the number of SP cells in human, rabbit, and rat limbal epithelia, 13 23 24 but corresponds to the number of SP cells found in the rat cornea. 23 Although it has been shown that the SP phenotype is associated with ABCG2 expression, 33 the corollary is not necessarily true (i.e., not all cells expressing ABCG2 exhibit the SP phenotype). The studies of Umemoto and coworkers in humans, 16 rabbits, 29 and rats 23 showed that although the number of cells exhibiting the SP phenotype was less than 2% in the limbus, immunochemistry revealed that a larger proportion (approximately 10%) of limbal basal epithelial cells expressed ABCG2 transporter. 23 Similarly, Budak et al. 18 suggested the existence of a significantly higher number of ABCG2+ cells than SP cells. This discrepancy was explained by the differences in the transport activity of ABCG2. Umemoto et al. 23 also showed that in the rat, unlike the human and rabbit, the central cornea contains cells with the SP phenotype but that these cells expressed significantly lower levels of putative SC markers than did the SP cells in the limbus. In addition, SP cells found in the rat cornea had a different profile on forward scatter analyses than SP cells in the limbus. Our study showed that mouse limbal cells with the SP phenotype from the light cell fraction of the Percoll gradient had distinctive light-scattering properties from SP cells from the dense cell fraction. It appears that the light cell fraction resembles the SP cells described by Umemoto et al. 23 in the rat central cornea rather than the basal limbal SCs. A high expression of the corneal epithelial cell differentiation marker K12 in the light cell population supports this analysis. It is therefore apparent that interspecies differences exist in the distribution and properties of corneal epithelial cells with limbal SC characteristics and that the mouse may, in this respect, represent a unique species different from human, rabbit, or rat. 
The limbal cells isolated in the dense cell fraction (80% Percoll) mimicked more closely the limbal SCs described in the human and rabbit by both SC characteristics (small cell size, expression of ABCG2, p63 and Lgr5, the SP phenotype) and by growth properties. These cells occurred in a quiescent state and did not proliferate within the first 3 days in tissue culture, as has been described for SP cells in the rabbit. 24 Budak et al. 18 showed that SP cells remained quiescent for at least 72 hours after seeding, whereas the non-SP cells began to divide within 24 to 48 hours. However, when we cultured the dense cell population on a 3T3 feeder cell monolayer, they exhibited a strong proliferative activity. Similarly, de Paiva et al. 13 showed that human limbal SP cells proliferate better on feeder cells than do non-SP cells. To evaluate growth properties of individual cell fractions, we cultured these cells at low cell concentrations. The cells from the light fraction formed colonies of fibroblast-like cells and their growth was enhanced in the presence of epidermal or fibroblast growth factor. On the contrary, the cells from the dense fraction did not grow in cultures without feeder cells, even in the presence of the growth factors. A similar pattern of proliferation and responsiveness to the growth factors was observed in the cells from the light fraction when cultured on a 3T3 cell monolayer. However, the cells from the dense fractions that did not proliferate in cultures without feeder cells, formed on the 3T3 cell monolayer colonies of spheric cells, and their growth was not significantly influenced by epidermal or fibroblast growth factor. It is possible that quiescent, slowly dividing limbal SCs (separated in the dense fraction) require a specific niche (feeder cells) to support their proliferation. 
This study showed that there are two distinct populations of corneal epithelial cells with SC characteristics (expression of ABCG2 and Lgr5, an SP phenotype) that can be isolated from the mouse limbus and that Percoll gradient centrifugation is a convenient method of enriching and harvesting such cells for the study of their characteristics, growth requirements, and use to treat various limbal SC deficiencies in experimental models. 
 
Table 1.
 
Mouse Primer Sequences Used for Real-Time PCR
Table 1.
 
Mouse Primer Sequences Used for Real-Time PCR
Gene Sense Primer Antisense Primer
GADPH GGG TGT GAA CCA CGA GAA AT ACA CAT TGG GGG TAG GAA CA
ABCG2 GCC TTG GAG TAC TTT GCA TCA AAA TCC GCA GGG TTG TTG TA
p63 TGG AAA ACA ATG CCC AGA CT CTG CTG GTC CAT GCT GTT C
Lgr5 CTT CAC TCG GTG CAG TGC T CAG CCA GCT ACC AAA TAG GTG
K12 CTG TGG AGG CCT CTT TTC TG ATT CCA GCT ATC CCC AAT CC
Figure 1.
 
Flow cytometry analysis (light-scattering profile) of freshly isolated, unseparated mouse limbal cells. Limbal tissue from normal BALB/c mice was trypsin-dissociated, and single cell suspensions were analyzed according to their size (FSC) and granularity (SSC) profiles. A representative dot-plot is shown.
Figure 1.
 
Flow cytometry analysis (light-scattering profile) of freshly isolated, unseparated mouse limbal cells. Limbal tissue from normal BALB/c mice was trypsin-dissociated, and single cell suspensions were analyzed according to their size (FSC) and granularity (SSC) profiles. A representative dot-plot is shown.
Table 2.
 
Recovery of Original Limbal Cells and the Proportion of Cells in Individual Fractions after Percoll Gradient Centrifugation
Table 2.
 
Recovery of Original Limbal Cells and the Proportion of Cells in Individual Fractions after Percoll Gradient Centrifugation
Exp. % of Original Cells in the Percoll Fraction Cell Recovery (%)
40 50 60 70 80 Bottom
1 14.2 17.2 16.3 22.8 7.2 8.0 85.7
2 10.4 17.3 27.1 13.5 3.1 8.4 79.8
3 13.0 11.6 23.1 17.3 7.7 9.7 82.4
4 10.2 13.3 22.1 21.7 6.1 4.2 77.6
5 14.6 16.3 20.0 20.3 7.1 8.4 86.7
Figure 2.
 
Expression of genes for SC markers ABCG2 (A), Lgr5 (B), and p63 (C), and for corneal differentiation marker K12 (D) in unseparated limbal cells and in individual fractions from a Percoll gradient. Real-time PCR was performed on unseparated limbal cells (total) and cells retained on 40%, 50%, 60%, 70%, and 80% gradients. Data are the mean ± SD of three separate experiments. The comparative Ct method was used to determine the change in targeted gene expression normalized by the internal control gene GAPDH. *P < 0.05, **P < 0.01.
Figure 2.
 
Expression of genes for SC markers ABCG2 (A), Lgr5 (B), and p63 (C), and for corneal differentiation marker K12 (D) in unseparated limbal cells and in individual fractions from a Percoll gradient. Real-time PCR was performed on unseparated limbal cells (total) and cells retained on 40%, 50%, 60%, 70%, and 80% gradients. Data are the mean ± SD of three separate experiments. The comparative Ct method was used to determine the change in targeted gene expression normalized by the internal control gene GAPDH. *P < 0.05, **P < 0.01.
Figure 3.
 
SP profile of freshly isolated unseparated mouse limbal cells and cells from the Percoll gradient fractions. Unseparated limbal cells (A, B) or cells from fraction 40% (C, D), 60% (E, F), or 80% (G, H) gradients were subjected to Hoechst 33342 exclusion assay. The cells were analyzed by flow cytometry (AH), and the dye efflux from the SP was blocked by verapamil (B, D, F, H).
Figure 3.
 
SP profile of freshly isolated unseparated mouse limbal cells and cells from the Percoll gradient fractions. Unseparated limbal cells (A, B) or cells from fraction 40% (C, D), 60% (E, F), or 80% (G, H) gradients were subjected to Hoechst 33342 exclusion assay. The cells were analyzed by flow cytometry (AH), and the dye efflux from the SP was blocked by verapamil (B, D, F, H).
Figure 4.
 
Light-scattering profile of SP cells from unseparated freshly isolated mouse limbal cells (A), or from the top (40% Percoll) (B) and the bottom (80% Percoll) (C) fraction. Shown are the light-scattering properties of cells denoted by each enclosed area in Figures 3A 3C and 3G .
Figure 4.
 
Light-scattering profile of SP cells from unseparated freshly isolated mouse limbal cells (A), or from the top (40% Percoll) (B) and the bottom (80% Percoll) (C) fraction. Shown are the light-scattering properties of cells denoted by each enclosed area in Figures 3A 3C and 3G .
Figure 5.
 
Growth properties of unseparated, freshly dissociated mouse limbal cells and cells from individual fractions obtained after Percoll gradient centrifugation. Unseparated limbal cells (total cell population) or cells retained on 40%, 50%, 60%, 70%, and 80% Percoll gradient fractions were seeded into wells of 96-well tissue culture plates (5,000 cells/well) (A) or into wells containing a 3T3 fibroblast feeder layer (B). Cell proliferation was determined by incorporation of [3H]thymidine added to the cultures for the last 6 hours of the 96-hour incubation period. The results are expressed by the counts per minute of incorporated [3H]thymidine (A) or as the increase in the proliferation of particular cell population cultivated on the 3T3 feeder layer in comparison to proliferation of the same cells cultivated without the 3T3 feeder cells. Each bar represents the mean ± SE of results in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from the control (unseparated limbal cells).
Figure 5.
 
Growth properties of unseparated, freshly dissociated mouse limbal cells and cells from individual fractions obtained after Percoll gradient centrifugation. Unseparated limbal cells (total cell population) or cells retained on 40%, 50%, 60%, 70%, and 80% Percoll gradient fractions were seeded into wells of 96-well tissue culture plates (5,000 cells/well) (A) or into wells containing a 3T3 fibroblast feeder layer (B). Cell proliferation was determined by incorporation of [3H]thymidine added to the cultures for the last 6 hours of the 96-hour incubation period. The results are expressed by the counts per minute of incorporated [3H]thymidine (A) or as the increase in the proliferation of particular cell population cultivated on the 3T3 feeder layer in comparison to proliferation of the same cells cultivated without the 3T3 feeder cells. Each bar represents the mean ± SE of results in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from the control (unseparated limbal cells).
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Figure 1.
 
Flow cytometry analysis (light-scattering profile) of freshly isolated, unseparated mouse limbal cells. Limbal tissue from normal BALB/c mice was trypsin-dissociated, and single cell suspensions were analyzed according to their size (FSC) and granularity (SSC) profiles. A representative dot-plot is shown.
Figure 1.
 
Flow cytometry analysis (light-scattering profile) of freshly isolated, unseparated mouse limbal cells. Limbal tissue from normal BALB/c mice was trypsin-dissociated, and single cell suspensions were analyzed according to their size (FSC) and granularity (SSC) profiles. A representative dot-plot is shown.
Figure 2.
 
Expression of genes for SC markers ABCG2 (A), Lgr5 (B), and p63 (C), and for corneal differentiation marker K12 (D) in unseparated limbal cells and in individual fractions from a Percoll gradient. Real-time PCR was performed on unseparated limbal cells (total) and cells retained on 40%, 50%, 60%, 70%, and 80% gradients. Data are the mean ± SD of three separate experiments. The comparative Ct method was used to determine the change in targeted gene expression normalized by the internal control gene GAPDH. *P < 0.05, **P < 0.01.
Figure 2.
 
Expression of genes for SC markers ABCG2 (A), Lgr5 (B), and p63 (C), and for corneal differentiation marker K12 (D) in unseparated limbal cells and in individual fractions from a Percoll gradient. Real-time PCR was performed on unseparated limbal cells (total) and cells retained on 40%, 50%, 60%, 70%, and 80% gradients. Data are the mean ± SD of three separate experiments. The comparative Ct method was used to determine the change in targeted gene expression normalized by the internal control gene GAPDH. *P < 0.05, **P < 0.01.
Figure 3.
 
SP profile of freshly isolated unseparated mouse limbal cells and cells from the Percoll gradient fractions. Unseparated limbal cells (A, B) or cells from fraction 40% (C, D), 60% (E, F), or 80% (G, H) gradients were subjected to Hoechst 33342 exclusion assay. The cells were analyzed by flow cytometry (AH), and the dye efflux from the SP was blocked by verapamil (B, D, F, H).
Figure 3.
 
SP profile of freshly isolated unseparated mouse limbal cells and cells from the Percoll gradient fractions. Unseparated limbal cells (A, B) or cells from fraction 40% (C, D), 60% (E, F), or 80% (G, H) gradients were subjected to Hoechst 33342 exclusion assay. The cells were analyzed by flow cytometry (AH), and the dye efflux from the SP was blocked by verapamil (B, D, F, H).
Figure 4.
 
Light-scattering profile of SP cells from unseparated freshly isolated mouse limbal cells (A), or from the top (40% Percoll) (B) and the bottom (80% Percoll) (C) fraction. Shown are the light-scattering properties of cells denoted by each enclosed area in Figures 3A 3C and 3G .
Figure 4.
 
Light-scattering profile of SP cells from unseparated freshly isolated mouse limbal cells (A), or from the top (40% Percoll) (B) and the bottom (80% Percoll) (C) fraction. Shown are the light-scattering properties of cells denoted by each enclosed area in Figures 3A 3C and 3G .
Figure 5.
 
Growth properties of unseparated, freshly dissociated mouse limbal cells and cells from individual fractions obtained after Percoll gradient centrifugation. Unseparated limbal cells (total cell population) or cells retained on 40%, 50%, 60%, 70%, and 80% Percoll gradient fractions were seeded into wells of 96-well tissue culture plates (5,000 cells/well) (A) or into wells containing a 3T3 fibroblast feeder layer (B). Cell proliferation was determined by incorporation of [3H]thymidine added to the cultures for the last 6 hours of the 96-hour incubation period. The results are expressed by the counts per minute of incorporated [3H]thymidine (A) or as the increase in the proliferation of particular cell population cultivated on the 3T3 feeder layer in comparison to proliferation of the same cells cultivated without the 3T3 feeder cells. Each bar represents the mean ± SE of results in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from the control (unseparated limbal cells).
Figure 5.
 
Growth properties of unseparated, freshly dissociated mouse limbal cells and cells from individual fractions obtained after Percoll gradient centrifugation. Unseparated limbal cells (total cell population) or cells retained on 40%, 50%, 60%, 70%, and 80% Percoll gradient fractions were seeded into wells of 96-well tissue culture plates (5,000 cells/well) (A) or into wells containing a 3T3 fibroblast feeder layer (B). Cell proliferation was determined by incorporation of [3H]thymidine added to the cultures for the last 6 hours of the 96-hour incubation period. The results are expressed by the counts per minute of incorporated [3H]thymidine (A) or as the increase in the proliferation of particular cell population cultivated on the 3T3 feeder layer in comparison to proliferation of the same cells cultivated without the 3T3 feeder cells. Each bar represents the mean ± SE of results in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from the control (unseparated limbal cells).
Table 1.
 
Mouse Primer Sequences Used for Real-Time PCR
Table 1.
 
Mouse Primer Sequences Used for Real-Time PCR
Gene Sense Primer Antisense Primer
GADPH GGG TGT GAA CCA CGA GAA AT ACA CAT TGG GGG TAG GAA CA
ABCG2 GCC TTG GAG TAC TTT GCA TCA AAA TCC GCA GGG TTG TTG TA
p63 TGG AAA ACA ATG CCC AGA CT CTG CTG GTC CAT GCT GTT C
Lgr5 CTT CAC TCG GTG CAG TGC T CAG CCA GCT ACC AAA TAG GTG
K12 CTG TGG AGG CCT CTT TTC TG ATT CCA GCT ATC CCC AAT CC
Table 2.
 
Recovery of Original Limbal Cells and the Proportion of Cells in Individual Fractions after Percoll Gradient Centrifugation
Table 2.
 
Recovery of Original Limbal Cells and the Proportion of Cells in Individual Fractions after Percoll Gradient Centrifugation
Exp. % of Original Cells in the Percoll Fraction Cell Recovery (%)
40 50 60 70 80 Bottom
1 14.2 17.2 16.3 22.8 7.2 8.0 85.7
2 10.4 17.3 27.1 13.5 3.1 8.4 79.8
3 13.0 11.6 23.1 17.3 7.7 9.7 82.4
4 10.2 13.3 22.1 21.7 6.1 4.2 77.6
5 14.6 16.3 20.0 20.3 7.1 8.4 86.7
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